ABSTRACT: The presence and expression of nitrogenase genes in diazotrophic prokaryotes has been documented in oligotrophic surface waters of the North Pacific gyre; however, there have been few investigations in other ocean basins. We initiated a study into the ecology of planktonic diazotrophs at 4 stations in the Sargasso Sea. Nucleic acid samples from the surface to 5890 m were collected on several dates between October 2003 and September 2005. Surprisingly, nifH could only be amplified from nano- and picoplankton DNA in surface waters on only 1 of 2 sampling dates at 1 station in the Sargasso Sea. Although no nifH was detected in the surface DNA samples earlier in the year at that station, Crocosphaera sp. cyanobacterial nifH was detected in enrichment cultures initiated with surface water collected at the time. In contrast to surface waters, diazotrophs were more consistently detected in deeper waters, including at several stations elsewhere globally. The nifH microarray fingerprints from the Sargasso Sea from 0, 5 and 25 m depths were remarkably consistent between samples taken 2 yr apart, but were different from fingerprints from subeuphotic waters. The results of our study suggest the presence of diazotrophic plankton in the Sargasso Sea, which is not constrained to surface waters. Furthermore, our study demonstrates that a deep-water diazotrophic flora persists in the meso- and abyssopelagic zones in the presence of high concentrations of combined nitrogen; however, the gene expression patterns of such species and their role in the N cycle remain unknown.